Compositions, methods and kits for enhancing PCR specificity

Abstract

The present disclosure provides novel primers and method for the detection of specific nucleic acid sequences. The primers and methods provided herein are useful in a wide variety of molecular biology applications and are particularly useful in allele-specific PCR.

What is claimed is: 
     
       1. A composition comprising:
 a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a sequence-targeted amplification restrictive (STAR) tag sequence which is the same as all or a portion of a target nucleic acid hybridization sequence of the second primer; and 
 a detectable label attached to the first oligonucleotide primer, the second oligonucleotide primer, or a probe, 
 wherein the STAR tag sequence further comprises a portion of an internal amplicon sequence of a target nucleic acid. 
 
     
     
       2. The composition of  claim 1 , wherein the sequence shared between the first and the second primers is between about 3 and about 15 nucleotides in length. 
     
     
       3. The composition of  claim 2 , wherein the first primer and the second primer are a pair of forward and reverse amplification primers. 
     
     
       4. A polynucleotide extension product comprising:
 a sequence-targeted amplification restrictive (STAR) tag sequence at the 5′ end and a sequence complementary to the STAR tag sequence to the 3′ side of the STAR tag sequence; and 
 a detectable label attached to the polynucleotide extension product, 
 wherein the STAR tag sequence and the complementary sequence render the extension product capable of forming a stem-loop structure, 
 wherein the STAR tag sequence is part of a first primer and wherein the STAR tag sequence comprises a shared sequence the same as all or a portion of a second primer to be used with the first primer in an amplification reaction, and 
 wherein the STAR tag sequence and the second primer share a common sequence of about 3 to about 15 nucleotides. 
 
     
     
       5. A method of detecting a nucleic acid comprising:
 providing the composition of  claim 1 ; 
 hybridizing a target nucleic acid with the first oligonucleotide primer; 
 extending the oligonucleotide to form an extension product; 
 amplifying the extension product to form an amplification product; and 
 detecting the presence or absence of the amplification product, thereby detecting the target nucleic acid. 
 
     
     
       6. A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
 providing the composition of  claim 1 ; 
 hybridizing a target nucleic acid with the first oligonucleotide primer; 
 extending the oligonucleotide to form an extension product; 
 incubating the extension product with an amplification reaction mixture; and 
 detecting the presence or absence of the extension product. 
 
     
     
       7. A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
 providing the composition of  claim 1 ; 
 hybridizing a target nucleic acid with the first oligonucleotide primer; and 
 extending the oligonucleotide to form an extension product, wherein the extension product inhibits or substantially reduces undesired amplification of the target nucleic acid. 
 
     
     
       8. The composition of  claim 1 , wherein the first primer has a length of about 20 to about 60 nucleotides. 
     
     
       9. The composition of  claim 1 , wherein the STAR tag sequence is about 10 to about 40 nucleotides in length. 
     
     
       10. The composition of  claim 4 , wherein the STAR tag sequence is about 10 to about 40 nucleotides in length. 
     
     
       11. A composition comprising:
 a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a sequence-targeted amplification restrictive (STAR) tag sequence which is the same as all or a portion of a target nucleic acid hybridization sequence of the second primer; and 
 a detectable label attached to the first oligonucleotide primer, the second oligonucleotide primer, or a probe, 
 wherein the first primer has a length of about 20 to about 60 nucleotides. 
 
     
     
       12. The composition of  claim 11 , wherein the sequence shared between the first and the second primers is between about 3 and about 15 nucleotides in length. 
     
     
       13. The composition of  claim 12 , wherein the first primer and the second primer are a pair of forward and reverse amplification primers. 
     
     
       14. The composition of  claim 11 , wherein the STAR tag sequence is about 10 to about 40 nucleotides in length. 
     
     
       15. A composition comprising:
 a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a sequence-targeted amplification restrictive (STAR) tag sequence which is the same as all or a portion of a target nucleic acid hybridization sequence of the second primer; and 
 a detectable label attached to the first oligonucleotide primer, the second oligonucleotide primer, or a probe, 
 wherein the STAR tag sequence is about 10 to about 40 nucleotides in length. 
 
     
     
       16. The composition of  claim 15 , wherein the sequence shared between the first and the second primers is between about 3 and about 15 nucleotides in length. 
     
     
       17. The composition of  claim 16 , wherein the first primer and the second primer are a pair of forward and reverse amplification primers. 
     
     
       18. A polynucleotide extension product comprising:
 a sequence-targeted amplification restrictive (STAR) tag sequence at the 5′ end and a sequence complementary to the STAR tag sequence to the 3′ side of the STAR tag sequence; and 
 a detectable label attached to the polynucleotide extension product, 
 wherein the STAR tag sequence and the complementary sequence render the extension product capable of forming a stem-loop structure, 
 wherein the STAR tag sequence is about 10 to about 40 nucleotides in length.